Life for all aerobic organisms, including humans, depends on the activation of O2 by metals to provide selective and rapid oxidation of biological molecules. Metalloenzymes have evolved a variety of chemical pathways to efficiently utilize the oxidizing power of abundant O2. Mn and non-heme Fe dioxygenases catalyze the cleavage of the aromatic ring of the substrate with incorporation of both oxygen atoms from O2. This reaction is a key step in the ability of Nature to reclaim large quantities of carbon sequestered in aromatic compounds. Tryptophan 2,3-dioxygenase (TDO) catalyzes the insertion of dioxygen and oxidative cleavage of the indole ring of L-tryptophan (L-Trp), converting it to N-formylkynurenine (NFK). The kynurenine pathway constitutes the major route of de novo biosynthesis of NAD, one of the essential redox cofactors in all living systems. The alteration of intermediate metabolites of this pathway can lead to numerous physiological and pathological conditions, including: cataract formation, cerebral malaria, Alzheimer's disease, HIV infection, Huntington's disease and ischemic brain injury. TDO is responsible for oxidizing over 99% of the free L-Trp in intracellular and extracellular pools. In addition, the levels of tryptophan regulated by TDO can affect the synthesis of serotonin, a known neurotransmitter. These two classes of enzymes represent two such fundamental differences in how Nature has evolved strategies for efficient substrate oxidation. The active metal site of the Mn and non-heme Fe dioxygenases has available metal coordination sites for both the substrate and O2. In contrast, the heme containing TDO has only one available metal coordination site, which binds O2, and the substrate L-Trp binds to the protein in a pocket close, but away from the Fe. Biomimetic model complexes will be studied to both enhance our ability to interpret complicated electronic properties of biomolecules and to aid our understanding of the structural and chemical aspect of metalloenzymes that are important in catalysis. Our goal in this proposal is to provide insight into how these enzymes perform their function by studying the atomic level changes that occur in the metal active site as the enzymes turn over their natural substrate. It is anticipated that such studies will provide a better understanding of how Nature constructs enzymatic active sites to perform selective and efficient oxidation of substrates. This research is made possible by our advances in EPR methodology. We have created software for the interpretation of EPR spectra which allows an unprecedented ability to quantitatively characterize virtually all paramagnetic metal sites in proteins and enzymes.